Certain fungi, known as the deep or systemic fungi, are characterized by involvement of visceral organs or penetrating types of infection. Besides true fungi, actinomycetes and nocardiae are bacteria that produce disease resembling deep fungal infection in many ways. These are discussed in the chapter on bacterial infections. The systemic fungi include Blastomyces dermatitidis (blastomycosis), Coccidioides immitis (coccidioidomycosis), Cryptococcus neoformans (cryptococcosis), Histoplasma capsulatum (histoplasmosis), and Sporothrix schenckii (sporotrichosis). Certain Candida species (especially Candida albicans and Candida tropicalis), Aspergillus species (Aspergillus fumigatus and Aspergillus flavus), and certain zygomycetes (Rhizopus species and Mucor species) may, on occasion, produce infection that would qualify as a systemic mycosis. Blastomyces, Coccidioides, Histoplasma, and Sporothrix organisms are considered diphasic (dimorphic) fungi, since they grow as a mycelial phase in culture but in a yeast (budding) phase within tissue infections.

Diagnosis of Fungal Infections

The diagnosis of mycotic infection can be assisted in several ways:

1. Wet mount of scraping, exudate, fresh swab smear, or other specimen such as sputum; usually done with 10% potassium hydroxide (KOH). India ink or nigrosin preparations are used for cryptococcosis. The advantages of wet mounting are same-day results and, in some instances, reasonably accurate diagnosis. Disadvantages are that few laboratory personnel are expert in this technique, and consequently there are frequent false positive and negative results. A recent aid to wet-mount examination are naturally fluorescing compounds, such as Calcofluor white, that bind nonspecifically to fungus cell walls and can outline the organism when it is viewed with the proper filters under a fluorescent microscope. Unfortunately, false negative results are frequent regardless of technique because the specimen obtained may not contain organisms. Also, in most cases (except possibly cryptococcosis) the most the technique can offer is recognition that a mycotic infection may be present without reliable identification of what the organism is, or its species. Speciation may be important because some species are more likely to be true pathogens in certain body areas than other species. When material such as sputum is examined, there is often a problem in deciding whether an organism is causing infection, is colonizing the area without actual infection, or is a contaminant.
2. Stained smear of a clinical specimen. Gram stain or Papanicolaou stain can be used. Wright’s stain or Giemsa stain is used for histoplasmosis. The advantages of the stained smear are same-day results and a permanent preparation that may be a little easier to interpret than a wet-mount preparation. However, others find the wet preparation easier to examine. The disadvantages are the same as those of the wet-mount preparation.
3. Tissue biopsy with demonstration of the organism by special stains, such as periodic acid-Schiff or methenamine silver. The yield from this procedure depends on whether the biopsy specimen contains organisms, the number of organisms present, and whether the organism is suspected so that the special stains are actually used.
4. Culture of a lesion. This permits definite isolation of an organism with speciation. The yield depends on whether the proper specimen is obtained, whether the organism is still alive by the time it reaches the laboratory, whether proper culture media are used, and the experience of the technologists. Because of the locations involved in deep mycotic infections, it may be difficult to secure a specimen or obtain material from the correct area. The organisms usually take several days to grow.
5. Serologic tests. The major advantage is that specimens for other types of tests may not be available or the results may be negative. The disadvantages are the usual need for two specimens (“acute” and “convalescent”) and the long time period involved, the second specimen being obtained 1-2 weeks after the first to see if there is a rising titer. This means 2-3 weeks’ delay, possibly even more, since the specimens usually must be sent to a reference laboratory. There are usually a significant percentage of false negative results (sometimes a large percentage), and there may be a certain number of false positive and nondiagnostic results as well.
6. Skin tests. The advantage is a result in 24-48 hours. The disadvantages include the time period needed to develop antibodies, false positive or negative test results, and the problem of differentiating a positive result due to old infection from one due to recent or active infection. In addition, the skin test in some cases may induce abnormality in the serologic tests, so a rising titer does not have its usual significance.

Blastomycosis

Blastomycosis may involve primarily the skin or the visceral organs. Granulomatous lesions are produced that are somewhat similar histologically to the early lesions of tuberculosis. Skin test results are unreliable, reportedly being positive in only about 50% of cases. Complement fixation (CF) tests also detect fewer than 50% of cases and produce false positive results in Histoplasma infections. Immunodiffusion tests are more specific for blastomycosis and are reported to detect about 80% of active cases. These procedures usually must be sent to a reference laboratory, since the number of requests for these tests in most institutions is quite small.

Coccidioidomycosis

Coccidioidomycosis is most often contracted in the San Joaquin Valley of California but occasionally appears elsewhere in the Southwest. It has a predilection for the lungs and hilar lymph nodes but occasionally may become systemic to varying degrees. Clinical symptoms are most often pulmonary, manifested usually by mild or moderate respiratory symptoms and sometimes by fever of unknown origin. Rarely, overwhelming infection much like miliary tuberculosis develops. Diagnosis is usually made through either biopsy or serologic tests. The most sensitive tests are tube precipitin (results of which become positive 1-3 weeks after onset of infection, with about 80% of cases positive by 2 weeks), latex agglutination (LA; slightly more sensitive than tube precipitin, but with 6%-10% false positive results), and immunodiffusion. The tube precipitin test usually reverts to negative by 6 months after onset of infection. A CF test is also widely used, especially for spinal fluid specimens. In cerebrospinal fluid (CSF) specimens it detects more than 90% of active coccidioidomycosis infections, whereas the tube precipitin test is not reliable on spinal fluid. These tests usually must be sent to a reference laboratory unless the laboratory receiving the request is located in an area where coccidioidomycosis is endemic. The coccidioidomycosis skin test is very useful, being equally as sensitive as the serologic tests. It does not produce an antibody response that would interfere with the other tests.

Cryptococcosis

Cryptococcosis (torulosis) is a fungal disease with a marked predilection for lung and brain. Pigeon feces seems to be the major known source of human exposure. Persons with illnesses that are associated with decreased immunologic resistance, such as acquired immunodeficiency syndrome (AIDS), Hodgkin’s disease, and acute leukemia, or those undergoing therapy with steroids or immunosuppressive agents are particularly susceptible. Pulmonary infection is apparently more common than central nervous system (CNS) infection but is often subclinical. Pulmonary infection radiologically can present in much the same way as tuberculosis and histoplasmosis, such as pneumonia or focal nodules, or occasionally as military lesions. CNS system disease typically occurs without respiratory disease and typically is very slowly progressive but may occasionally be either asymptomatic or severe and acute. In CNS disease, headache is found in about 75% of cases and fever in about 35%. Peripheral blood complete blood cell count and erythrocyte sedimentation rate are most often normal in respiratory and CNS cryptococcosis. Laboratory findings in CNS disease are described in Chapter 19. Diagnosis can be made through culture of sputum or CSF, by histologic examination of biopsy specimens (special stains for fungi are required), by microscopic examination of CSF using an india ink or nigrosin preparation to show the characteristic organism thick capsule (Chapter 19), and by serologic tests. Serologic tests that detect either antigen or antibody are available. Antibody is usually not present in the CSF; and in serum, antibody appearance is inconsistent in the early acute phase of the illness. In addition, some of the antibody-detection systems cross-react with histoplasmosis antibody. Cryptococcus antigen detection systems do not react when the patient is infected by other fungi.

The most widely used serologic test is the slide latex agglutination procedure, which detects antigen. In localized pulmonary cryptococcosis, the LA test can be used on serum, but it detects fewer than 30% of cases. In patients with cryptococcal meningitis, the latex test can be used on either serum or CSF specimens. When used on CSF specimens, it detects about 85%-90% of culture-positive cases of cryptococcal meningitis (range, 75%-100%) versus 40%-50% for india ink. Testing both serum and CSF increases the number of LA-detectable cases. Rheumatoid factor may produce a false positive reaction; so patient serum must be heat inactivated, and a control for rheumatoid factor and nonspecific agglutinins must be used when either serum or CSF is tested. Some kits now incorporate pretreatment of the specimen with pronase, a proteolytic enzyme, to inactivate interfering substances. Occasional false positive results have been reported in patients with malignancy or collagen-vascular disease. False positive results also were reported due to culture medium contamination of the CSF specimen when a portion was removed by wire loop, plated on culture media, then the loop reintroduced into the CSF specimen to obtain fluid for LA testing. It should be mentioned that a few investigators have not obtained as good results on CSF specimens as most others have. Some of the test evaluations on CSF reported in the literature are difficult to interpret, however, since kits by different manufacturers apparently produce different results, and some investigators used their own reagents. As noted previously, the latex test may be nonreactive in low-grade chronic infections. The half-life of cryptococcal polysaccharide antigen is about 48 hours, so that once positive, the results of a test detecting antigen may remain positive for several days even if therapy is adequate. Besides LA, an enzyme-linked immunosorbent assay (ELISA) test that is a little more sensitive than the latex tests is commercially available.

Histoplasmosis

Histoplasmosis is the most common of the systemic fungal infections. It is most often encountered in the Mississippi Valley and Ohio Valley areas but may appear elsewhere. Certain birds, especially chickens and starlings, are the most frequent vectors in the United States. In endemic areas, 60% or more of infected persons are asymptomatic. The remainder have a variety of illness patterns, ranging from mild or severe, acute or chronic pulmonary forms, to disseminated infection.

Histoplasmosis begins with a small primary focus of lung infection much like the early lesion of pulmonary tuberculosis. Thereafter, the lesion may heal or progress or reinfection may occur. Mild acute pulmonary infection with influenza-like symptoms may develop. The illness lasts only a few days, and skin test results, cultures, and chest x-ray films are usually normal. More severe acute pulmonary involvement produces a syndrome resembling primary atypical pneumonia. Chest x-ray films may show hilar adenopathy and single or multiple pulmonary infiltrates. Results of histoplasmin skin tests and CF or latex agglutination tests are negative during the first 2-3 weeks of illness but then become positive. Sputum culture is sometimes positive but not often. Chronic pulmonary histoplasmosis resembles chronic pulmonary tuberculosis clinically. Cavitation sometimes develops. Results of skin tests and CF tests usually are positive by the time the disease is chronic. Sputum is the most accessible material for culture, although results are reportedly negative in 50%-60% of cases. In clinically inactive pulmonary disease, such as coin lesions, sputum culture is usually negative. Even if the organism is present in the specimen, it takes 3-4 weeks for growth and identification. Histoplasmosis is a localized pulmonary disease in the great majority of patients, so there is usually little help from cultures obtained outside the pulmonary area. In the small group that does have disseminated histoplasmosis, either acute or chronic, there is a range of symptoms from a febrile disease with lymphadenopathy and hepatosplenomegaly to a rapidly fatal illness closely resembling miliary tuberculosis. In disseminated (miliary) histoplasmosis, standard blood cultures are positive in 40%-70% of patients (probably 60%-80% with newer culture methods). Bone marrow aspiration is the diagnostic method of choice; it is useful both for cultures and for histologic diagnosis of the organisms within macrohages on Wright-stained smear or fungus stain on a marrow clot section. Occasionally lymph node biopsy may be helpful. If it is performed, a culture should also be taken from the node before it is placed in fixative. Bone marrow aspiration and liver or lymph node biopsy are not helpful in the usual forms of histoplasmosis, which are localized to the lungs.

The most commonly used diagnostic test in histoplasmosis is the CF test. Titers of 1:16 are considered suspicious, and 1:32 or more are strongly suggestive of histoplasmosis. Two types of CF test are available, based on mycelial antigen and yeast phase antigen. The test based on yeast phase antigen is considerably more sensitive than that based on mycelial antigen. Neither test result is likely to be positive in the early phase of acute infection. Later on (about 3-4 weeks after infection), results of the yeast antigen CF test become positive in 70%-85% of cases. Some additional cases may be detected with the mycelial CF antigen. About 3.5%-12% of clinically normal persons demonstrate positive results, usually (but not always) in titers less than 1:16. Thirty-five percent to 50% of patients with positive CF test results in the literature could not be confirmed as having true histoplasmosis infections. How many of these positive results were due to previous old infection or localized active infection without proof is not known. Because of the false positive and negative results, a fourfold (two-dilution level) rise in titer is much more significant than a single result, whether the single result is positive or negative. The CF test result may be negative in 30%-50% of patients with acute disseminated (miliary) histoplasmosis and when the patient has depressed immunologic defenses or is being treated with steroids.

LA tests are also available and are reported to be a little more sensitive than the CF tests. However, there are conflicting reports on their reliability, with one investigator unable to confirm 90% of the cases with positive results and other studies being more favorable. Differences in reagents may be a factor. ELISA serologic methods have been reported with sensitivity equal to or better than CF. DNA probe methods have also been reported.

Besides serologic tests, a skin test is available. Results of the skin test become positive about 2-3 weeks after infection and remain positive for life in 90% of persons. The skin test result is falsely negative in about 50% of patients with disseminated (miliary) histoplasmosis and is said to be negative in about 10% of patients with cavitary histoplasmosis. A (single) skin test result is difficult to interpret, whether it is positive (because of past exposure) or negative (because it may be too early for reaction to develop, or reaction may be suppressed by miliary disease, depressed immunologic status, or steroid therapy). Also, about 15% of patients develop a positive CF test result because of the skin test. The histoplasmin skin test reacts in about 30% of patients who actually have blastomycosis and in about 40% of those with coccidioidomycosis. For these reasons, routine use of the histoplasmin skin test is not recommended.

In serious localized infection or widespread dissemination of the deep fungi, there is often a normocytic-normochromic or slightly hypochromic anemia. The anemia is usually mild or moderate in localized infection. In acute disseminated histoplasmosis, various cytopenias (or pancytopenia) are present in 60%-80% of cases, especially in infants.

Sporotrichosis

Sporotrichosis is caused by S. schenckii, which lives in soil and decaying plant material. Most of those who contract the disease are gardeners, florists, or farmers. The fungus is acquired through a scratch or puncture wound. The lymphocutaneous form constitutes two thirds to three fourths of all cases. A small ulcerated papule develops at the site of inoculation and similar lesions appear along lymphoid channels draining the original lesion area. Lymph nodes are not involved. The classic case is a person who does gardening and has contact with roses who develops a small ulceration on one arm followed by others in a linear ascending distribution. In children it is found as frequently on the body or face as on the extremities. Other than the lesions the patient usually has few symptoms.

The major diagnostic tests are culture of the lesions, biopsy, and serologic tests. The LA test, tube agglutination, and immunofluorescent test on serum are the most sensitive (±90% detection rate). CF tests detect approximately 65% of cases.